WO2013026947A1 - Light-controlled activation and internalisation of small molecules that can bind to double-stranded dna - Google Patents

Abstract

The invention relates to compounds formed by a small molecule that can bind to double-stranded DNA, covalently linked to a photolabile protecting group, and to the uses thereof. The deprotection of these compounds by means of light irradiation allows the activation of the aforementioned DNA-bound molecules to be controlled in space and time.

Description

 INTERNALIZATION AND PHOTOCONTROLLED ACTIVATION OF SMALL CAPABLE MOLECULES TO JOIN DOUBLE DNA

HEBRA FIELD OF THE INVENTION

The present invention relates to compounds that allow the spatially and temporarily controlled activation of small double stranded DNA binding molecules, as well as their uses.

BACKGROUND OF THE INVENTION The DNA molecule is involved in numerous basic processes for cell life and is the therapeutic target with which numerous drugs interact.

Through this interaction, physical-chemical changes in DNA occur that can have important consequences in the various activities in which the DNA molecule intervenes. As a result, compounds capable of binding to DNA are very useful tools in both diagnostic and therapeutic fields.

These DNA binding molecules can cause activation, modulation or inhibition of DNA function. They usually act causing the inhibition of basic processes for cell survival such as transcription or replication, causing cell death. Therefore, these types of molecules have been used in chemotherapy and for the treatment of bacterial, viral, fungal and parasitic diseases, among others. In addition, DNA is one of the most common targets of many of the anticancer drugs used in chemotherapy.

However, some of these molecules have a low specificity of cellular tissue, attacking not only the cancer cell or the microorganism but also healthy cells or host cells, respectively.

In order to reduce the high toxicity involved in the use of DNA-binding molecules, there is a need to develop methods that effectively control the activity of said molecules, both in terms of space and time. Current Opinion in Chemical Biology 2009, 13, 678-686 and Organic & Biomolecular Chemistry 2007, 5, 999-1005 documents describe the use of light-sensitive protecting groups to control the activation of biological molecules, as expression inducers gene, oligonucleotides or proteins. Tetrahedron Letters 2003, 44, 7307-7309, describes a derivative of Erenil or which, after irradiation with light, breaks down into 4-amidino-N- (3- hydroxypropyl) aniline and the cation 4-amidinobenzenodiazonio. The latter could cause DNA breakage. The Berenyl derivative described herein is capable of interacting with DNA and, therefore, cannot be used to control the inactivation and subsequent activation at the desired place and time of the diazonium salt 4-amidinobenzenodiazonium.

BRIEF DESCRIPTION OF THE INVENTION

The inventors of the present invention have discovered that the use of photolabile protecting groups allows temporarily inactivating the activity of small molecules that selectively interact with double stranded DNA. The resulting protected compound is not capable of binding to the double stranded DNA, whereby it is possible to have a non-active derivative that can be comfortably handled, and that is released at the precise time and place where it is desired to act by irradiation with light, thus inducing the interaction of the small molecule with the DNA in a controlled manner.

In this way, it is possible to activate the small double stranded DNA binding molecules efficiently and in a controlled manner.

Therefore, in a first aspect, the invention is directed to a compound formed by a small double stranded DNA binding molecule, covalently conjugated to a photolabile protecting group, with the proviso that said compound is not itself a molecule double stranded DNA binding.

In another aspect, the invention is directed to the use of a compound formed by a small double-stranded DNA binding molecule covalently linked to a photolabile protecting group, for in vitro control of gene expression and / or protein synthesis. These small molecules that bind DNA cause a change in the structure and / or functionality of the double stranded DNA molecule when they interact with it, preventing basic processes for cell survival and proliferation, such as transcription and replication. Therefore, this type of small double stranded DNA binding molecules have important applications such as antitumor, antibacterial, antiviral, antifungal and antiparasitic, among others.

Therefore, in another aspect the invention relates to the use of a compound formed by a small double stranded DNA binding molecule covalently linked to a photolabile protecting group, for the preparation of a medicament. This medication may be a controlled action medication, that is, a medication whose activity can be activated in a controlled manner at the desired time and / or place by irradiation with light.

In a further aspect, the present invention relates to the use of a compound formed by a small double-stranded DNA binding molecule covalently linked to a photolabile protecting group, for the preparation of a medicament for the treatment of proliferative diseases or diseases. microbial

In another aspect, the present invention also provides a pharmaceutical composition comprising at least one compound formed by a small double-stranded DNA binding molecule covalently linked to a photolabile protecting group, and at least one pharmaceutically acceptable excipient.

On the other hand, the spectral characteristics of some small molecules that bind double-stranded DNA change when binding to DNA and, therefore, are useful as DNA markers or dyes.

Therefore, the invention also relates to the use of a compound formed by a small double-stranded DNA binding molecule covalently linked to a photolabile protecting group, to mark in vitro specific areas of DNA and / or cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Left: the solid line represents the HPLC spectrum of protected compound © 1 in 20 mM Tris-HCl buffer solution; 100 mM NaCl; pH 7.5; The dashed line represents the HPLC spectrum of the same solution after irradiating for 15 min with UV light. Right: fluorescence emission spectrum at increase the irradiation time of a 0.5 μΜ solution of © 1 in 50 mM HEPES buffer, 100 mM NaCl in the presence of the hairpin oligonucleotide (containing the AAATTT target sequence (5 μΜ). Fork sequence AAATTT: 5'- GGCG AAA TTT CGC TTTTT GCG AAA TTT CGCC-3 '(SEQ ID NO: 1). Figure 2. Above: fluorescence emission spectrum of a 0.8 μΜ solution of © 2 in 50 mM Tris-HCl buffer, 100 mM NaCl, pH 7.5 with increasing irradiation time. Below: fluorescence emission microscopy of cells belonging to the Vero cell line (12.5 μΜ © 2 after 15 min incubation); A: before irradiation and deprotection, B: after deprotection by irradiation with UV light for 10 min, C: control experiment with 12.5 μΜ of 2 after 15 min. of incubation

Figure 3. Above: fluorescence microscopy of cells belonging to the Vero line incubated with compound © 2, after increasing irradiation times 365 nm). Below: A) Culture plate where cells are forming a monolayer before irradiation; B) same culture after irradiation through a cardboard mold. These photos were taken on the irradiated samples with a transilluminator.

Figure 4. Left: Fluorescence microscopy of Vero cells incubated with 50 μΜ of © 3 showing deprotection and redistribution of fluorescence from the nuclear membrane to the nucleus (t = 0 image with white light and fluorescence, t = 4 min until t = 16 min fluorescence images). Right: Schematic representation of the proposed activation dynamics of compound © 3.

Figure 5. Control of the effect of UV irradiation on ethidium bromide. A: 50 microM ethidium bromide before being irradiated. B: after being irradiated, where it is clearly observed that it does not suffer any variation and occurs in the cell nucleus. Figure 6. Control of the effect of UV irradiation on © 2 (Nvoc ₂ -ethidium) 50 microM. A: image with white light. B: before irradiation. C: after 15 minutes of irradiation. It is observed that © 2 is not visible with the microscope's optical cubes that cover red; Only after irradiation are the cells visible.

Figure 7. Control of the effect of UV irradiation on DAPI. A: DAPI 50 microM before being irradiated. B: after being irradiated no change is observed. Figure 8. Control of the effect of UV irradiation on © 3 (Nvoc ₂ -DAPI) 50 microM. A: image with white light. B: before irradiation. C: after 15 minutes of irradiation. It is noted that © 3 is not visible with the microscope's optical cubes that span blue; Only after irradiation are the cells visible. Figure 9. Antimicrobial activity of compound © 4 measured by disk diffusion of Candida albicans. (a) sterile disc with compound © 4 (without irradiation); (b) positive control with aza-pentamidine; (c) compound © 4 irradiated with UV light; (d) commercial antibiotic (clotrimazole); (e) negative control (buffer); (f) negative control irradiated with UV light. DETAILED DESCRIPTION OF THE INVENTION

Small double stranded DNA binding molecules

According to the first aspect mentioned above, the present invention relates to a compound formed by a small double-stranded DNA binding molecule that is covalently bound to a photolabile protecting group, with the proviso that said compound is not in turn a double stranded DNA binding molecule.

The objective of the present invention is to control the ability of small molecules to bind double stranded DNA. To do this, photolabile protective groups are used that inactivate the ability of the molecule to bind to DNA. Therefore, it is necessary that the compound of the invention, that is, the small double stranded DNA-binding molecule covalently linked to a photolabile protecting group, is not itself a molecule capable of binding to DNA. In this way it is possible to direct the inactive compound to the desired location for the subsequent release of the DNA binding molecule in a controlled manner.

The term "small double stranded DNA binding molecule" or "small molecule capable of binding double stranded DNA" refers, according to the present invention, to a molecule of molecular weight less than 300 Da which is capable of interacting with Double stranded DNA. Such molecules are well known in the state of the art (eg, SM Nelson, LR Ferguson, WA Denny, Non-covalent link d / DNA interactions: Minor groove binding agents. Mutation Res. 2007, 623, 24-40; "Small molecule DNA andRNA binders", Wiley-VCH 2003). In addition, the interaction of a molecule with DNA produces physical-chemical changes in it, which has given instead of various experimental techniques that allow to determine if a molecule interacts with DNA and also distinguish the different types of molecule-DNA interaction {Oncology, 2004, 27 (2), 69-79), such as by UV / visible spectrophotometry, mass spectrometry, nuclear magnetic resonance spectroscopy, calorimetric methods, circular dichroism, viscosimetry, etc.

There are different modes of interaction between small molecules and double stranded DNA. Depending on the type of interaction, a distinction is made between covalent junction (covalent type junctions between the molecule and DNA are formed) or non-covalent (such as hydrogen bonds or electrostatic interactions). Within the molecules that covalently bind to DNA are alkylating agents. Among the molecules that bind to DNA in a non-covalent way are molecules that bind to the grooves (major or minor) of DNA and intercalating agents.

Therefore, in one embodiment of the invention, the small double stranded DNA binding molecule is selected from molecules that bind to double stranded DNA covalently and molecules that bind noncovalently. Preferably, said molecule is selected from an alkylating agent, an intercalating agent and a groove binding molecule, preferably minor groove binding.

In the present invention, the term "alkylating agent" refers to molecules that bind to DNA covalently. This term includes both classic alkylating agents in which alkyl groups of these agents bind to the DNA molecule, such as compounds that do not have an alkyl group but also covalently bind to DNA, for example generating bonds between two chains of DNA (cross-link). In general, they are molecules with reactive electrophilic groups that covalently bind to DNA preferably through the nitrogen bases.

Alkylating agents are well known in the state of the art (eg, L. Kelland, The resurgence of platinum-based cancer chemotherapy, Nature Rev. Cancer 2007, 7, 573-584; T. Bando, H. Sugiyama, Synthesis and biological properties of sequence-specific DNA-alkylating pyrrole-imidazole polyamides, Acc. Chem. Res. 2006, 39, 935-944) Some typical alkylating agents include, for example, nitrosoureas (eg carmustine, lomustine, semustine, fotemustine, streptozocin) , nitrogen mustards (bischloroethylamines, eg chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, uracil mustard, trophosphamide, estramustine), aziridines (eg thiotepa, mitomycin C), alkylsulfonates (eg busulfan, clomesone), hydrazines and triazines (eg altretamine, dacarbazine, procarbazine complexes, procarbazine, pharmozine complexes no (eg cisplatin, carboplatin, oxaliplatin, nedaplatin, saltraplatin, lobaplatin).

In the present invention, the term "intercalating agent" refers to molecules that bind to double-stranded DNA in a non-covalent manner when intercalated between the nitrogen bases of the DNA. In general, they are flat polycyclic structures that interact with DNA through stacking or hydrophobic interactions.

Intercalating agents are well known in the state of the art (eg, L. Strekowski, B. Wilson, Noncovalent interactions with DNA: An overview. Mutation Res. 2007, 623, 3-13; JB Chaires, Drug-DNA Interactions, Curr. Opin. Struct. Biol. 1998, 8, 314-320) Some typical intercalating agents include, for example, anthracyclines (eg doxorubicin, daunorubicin, rubidazone, epirubicin, aclarubicin), acridines (eg acridine orange, proflavin, amsacrine , quinacrine), anthraquinones (eg mitoxantrone), naphthalimides (eg amonafide, elinafide), ethidium bromide, propidium iodide, Actinomycin D, quinine, mefloquine, ellipticin and dithercalin.

In the present invention, the term "groove binding molecule" refers to a molecule that binds within the groove of a double stranded DNA, preferably within the minor groove, through non-covalent junctions. More preferably, it is a molecule that binds to the minor groove of the double-stranded DNA through sequences rich in A and T.

The minor groove binding molecules are well known in the state of the art (eg, C. Bailly, JB Chaires, Sequence-specific DNA minor groove binders. Design and synthesis of netropsin and distamycin analogues, Bioconjugate Chem. 1998, 9, 513-538; DE Wemmer, Designed Sequence-Specific Minor Groove Ligands, Annu. Rev. Biophys. Biomol. Struct. 2000, 29, 439-461). Some typical minor groove binding molecules include, for example, diarylamine dynes (eg pentamidine, propamidine, aza-propamidine, aza-pentamidine, furamidine, stilbamidine, berenyl, DAPI (4 ', 6- diamidino-2-phenylindole)), netropsin, distamycin, lexithropsin, mitramycin, cromomycin A ₃ , olivomycin, antramycin, sibiromycin, bis-benzimidazoles (eg Hoechst 33258, Hoechst 33342).

In one embodiment of the invention, the small double stranded DNA binding molecule is selected from a DNA dye, an antitumor agent and an antimicrobial agent.

According to a particular embodiment, the small double stranded DNA binding molecule is a DNA dye. Preferably, the DNA dye is selected from acridine orange, DAPI, ethidium bromide, propionium iodide Hoechst 33258 and Hoechst 33342. According to another particular embodiment, the small molecule binding to the

Double stranded DNA is an antitumor agent. Preferably, the antitumor agent is selected from among alkylating agents of DNA, such as nitrosoureas (eg carmustine, lomustine, semustine, photemustine, streptozocin), nitrogenous mustards (bischlorethylamines, eg chlorambucil, cyclophosphamide, ifosfamide, mechlorethatamine, melchlophthatamine, melchlophthatamine, melostataamide , estramustine), aziridines (eg thiotepa, mitomycin C), alkylsulfonates (eg busulfan, clomesone), hydrazines and triazines (eg altretamine, dacarbazine, procarbazine, temozolamide) and platinum complexes (eg cisplatin, carboplatin, oxaliplatin, neplaplatin, neplatin lobaplatin); anthracyclines (e.g. doxorubicin, daunorubicin, rubidazone, epirubicin, aclarubicin); acridines (e.g. acridine orange, proflavin, amsacrine, quinacrine); anthraquinones (e. g. mitoxantrone); naphthalimides (e.g. amonafide, elinafide); Actinomycin D; ellipticin; ditercalinium; netropsin; and bis-benzimidazoles (e.g. Hoechst 33258, Hoechst 33342).

According to another particular embodiment, the small double strand DNA binding molecule is an antimicrobial agent. Preferably, the antimicrobial agent is selected from acridines (eg acridine orange, proflavin, amsacrine, quinacrine), quinine, mefloquine, diarylamidines (e.g., pentamidine, propamidine, aza-propamidine, aza-pentamidine, furamidine, strebamidine, berenyl , DAPI), netropsin. In one embodiment of the invention, the small double stranded DNA binding molecule is a compound of formula (la) or (Ib): HN NH

-X— B— NH ₂

H ₂ N NH ₂

 (the) (Ib)

 where:

 A and B are each independently selected from optionally substituted aryl and heteroaryl;

X may not exist, so that A and B are condensed, or it represents a single bond, -O-, -S-, -NH-, -OYO-, -SYS-, -NH-Y-NH-, or an optionally substituted Ci-C ₆ alkyl, aryl or heteroaryl;

Y is selected from optionally substituted Ci-C ₈ alkyl, aryl and heteroaryl, or a salt or solvate thereof.

Preferably, A and B are independently selected from among phenyl, indole or together form a phenanthridine ring, optionally substituted, preferably by C ₁ -C ₃ alkyl or C ₆ -C aryl _. In a preferred embodiment, A and B are independently selected from among phenyl, indole, or together represent a 5-ethyl-6- phenylphenanthridinium ring.

Preferably, X is selected from a bond, -OYO- and -NH-Y-NH-; where Y preferably represents a Ci-C ₈ alkyl group, more preferably a pentyl group.

In a preferred embodiment, the small double stranded DNA binding molecule is selected from pentamidine, aza-pentamidine, ethidium bromide, 4 ', 6- diamidino-2-phenylindole (DAPI), or a salt or solvate thereof .

 aza-pentamidine (1) ethidium bromide (2)

Photolabile protective groups In the present invention, the term "photolabile protecting group" is defined as a protecting group whose binding to a molecule is broken or released by exposure to light of an appropriate wavelength. Photolabile protecting groups, as well as the conditions for their preparation and subsequent deprotection, are known in the prior art (eg PJ Kocienski, "Protecting Groups", Thieme 2005) and include, for example, o-nitrobenzyl derivatives, derivatives of benzoin, phenacil derivatives, etc.

Preferably, in the present invention the photolabile protecting group is deprotected by irradiation with UV light (preferably of a wavelength between 200 and 400 nm, more preferably> 365 nm), preferably with a power of between 5 and 10 W, more preferably 8 W.

In a particular embodiment, the photolabile protecting group has the following structure:

 (Π)

 where

 n is selected from 0, 1, 2, 3 and 4;

R ¹ and each R ² are independently selected from hydrogen, Ci-C ₆ haloalkyl Ci-C _6, aryl C ₆ -Ci5, 3- to 15 members and heterocycle 3-15 optionally substituted members, N0 _2, CN , halogen, -OR ', -SR', -S (0) R ', -S (0) ₂ R', -OS (0) ₂ R ', -N (R') (R "), -C (0) R ', -C (0) OR', -C (0) N (R ') (R "), - OC (0) R' and -N (R ') C (0) R"; wherein each R 'and R "is independently selected from hydrogen, Ci-C ₆ haloalkyl _Ci-C6, C3-C7 aryl , C ₆ -Ci5, 3- to 15 - membered heterocycle and 3-15 membered optionally substituted;

or two R ² groups form, together with the phenyl ring to which they are attached, a heterocycle group.

In a particular embodiment of the invention, n is selected from 0, 1 and 2. In another particular embodiment, R ¹ is selected from hydrogen, methyl, trifluoromethyl and carboxyl. In a further embodiment, R ² represents methoxy, or two contiguous R ² radicals form a group -0-CH ₂ -0-. According to a particular embodiment, n is selected from 0, 1 and 2; R is selected from hydrogen and carboxyl; and R ² represents methoxy.

 Additionally, the inventors have observed that the presence in the protective group of ionizable groups at physiological pH makes it possible to control even more effectively the inactivation of the small double stranded DNA binding molecule. In particular, it has been observed that when the Nmoc protective group is used, the carboxylic group is ionized as a carboxylate at physiological pH, causing electrostatic repulsions between the protected compound and the phosphate groups of the DNA, which makes it possible to control even more effectively than the Protected compound object of the present invention cannot bind to DNA.

Therefore, in a further embodiment, the photolabile protecting group has an ionizable functional group at physiological pH. Preferably, at least one of R ¹ and R ² comprises an ionizable group at physiological pH. More preferably, at least one of

R ¹ and R ² is a carboxyl group.

 In a further embodiment, the photolabile protecting group is selected from:

 In a further embodiment, the compound of the invention is selected from a compound of formula (Illa) and (Illb):

 (Illb)

where A, B, X, n, R ¹ and R ² are as previously defined, or a salt or solvate thereof. In another particular embodiment, the compound of the invention is selected from:

o una sal o solvato del mismo. Adicionalmente, los inventores han observado que el empleo de grupos protectores permite no sólo controlar la activación de la molécula pequeña de unión al ADN de doble hebra, sino también modular sus propiedades fisicoquímicas. Se ha observado, por ejemplo, que el uso de Nvoc como grupo protector fotolábil mejora las propiedades de internalización en la célula del bromuro de etidio. Es importante destacar que el bromuro de etidio, debido a su elevada polaridad, atraviesa muy difícilmente la membrana celular, lo que ha dificultado el uso de esta molécula en experimentos in vivo. El uso de los grupos protectores mencionados previamente permite solucionar este problema al aumentar la hidrofobicidad de la molécula.

or a salt or solvate thereof. Additionally, the inventors have observed that the use of protective groups allows not only to control the activation of the small double stranded DNA binding molecule, but also to modulate its physicochemical properties. It has been observed, for example, that the use of Nvoc as a photolabile protecting group improves the internalization properties in the ethidium bromide cell. It is important to note that ethidium bromide, due to its high polarity, crosses the cell membrane very difficult, which has made it difficult to use this molecule in in vivo experiments. The use of the protective groups mentioned above allows to solve this problem by increasing the hydrophobicity of the molecule.

Definitions The term "alkyl" refers to a straight or branched chain hydrocarbon radical consisting of carbon and hydrogen atoms, which does not contain any unsaturation, having one to eight (Ci-C ₈ ), one to six ( Ci-C ₆ ), or one to three (Ci- C ₃ ) carbon atoms, and that is attached to the rest of the molecule by a single bond. Examples of alkyl groups include, but are not limited to, alkyl groups such as methyl, ethyl, n-propyl, isopropyl, isobutyl, 3-methyl-l-pentyl, 4-methyl-l-pentyl, 3,3-dimethyl -l-butyl, t-butyl, n-pentyl, isopentyl and n-hexyl. The term "C3-C7 cycloalkyl" means a non-aromatic monocyclic or polycyclic ring, comprising carbon and hydrogen atoms. A cycloalkyl group may have one or more carbon-carbon double bonds in the ring as long as the ring is not aromatic. Examples of cycloalkyl groups include, but are not limited to, fully saturated cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl, and cyclic and bicyclic terpenes, and cycloalkenyl groups, such as cyclopropenyl, cyclobutenyl, cyclohentenyl, cyclohexenyl cycloheptenyl, and unsaturated cyclic and bicyclic terpenes. A cycloalkyl group may not be substituted or substituted with one or two suitable substituents. Preferably, the cycloalkyl group is a monocyclic ring or bicyclic ring.

The term "heterocycle" refers to a non-aromatic monocyclic or polycyclic ring comprising carbon and hydrogen atoms and at least one heteroatom, preferably, from 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur and which may include condensed ring systems . A heterocycle group may be totally or partially saturated, or it may be aromatic (heteroaryl). Preferably, the heterocycle group is a monocyclic, bicyclic or tricyclic ring comprising from 3 to 15, preferably from 5 to 15, more preferably from 5 to 10 members and from 1 to 3 heteroatoms. Examples of heterocycle groups include aziridinyl, pyrrolidinyl, pyrrolidino, piperidinyl, piperidino, piperazinyl, piperazino, morpholinyl, morpholino, thiomorpholinyl, thiomorpholino, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, benzipyridine, pyridine pyridine, pyridine, pyridine, pyridine, pyridine pyridine pyrimidine, isothiazole, isoxazole, imidazole, indole, purine, quinoline, thiadiazole. A heterocycloalkyl group.

The term "halogen" refers to chlorine, bromine, iodine or fluorine. The term "aryl" refers to an aromatic group having between 6 and 15, preferably between 6 and 10 carbon atoms, comprising 1, 2 or 3 nuclei aromatic, optionally condensed, including the following non-limiting emplos: phenyl, naphthyl, diphenyl, indenyl, phenanthryl.

The term "haloalkyl" refers to an alkyl group as previously defined, where at least one hydrogen atom has been replaced by a halogen. Preferably, the term "haloalkyl" refers to a CF ₃ group.

As understood in this technical area, there may be a certain degree of substitution in the radicals defined above. References herein with respect to substituted groups indicate that the specified radical may be substituted at one or more positions available with one or more substituents. Such substituents include, for example and in a nonlimiting sense, Ci- ₆ alkyl, _C3-7 cicloal chyle, aryl, heterocycle, heteroaryl, halogen, cyano, nitro, trifluoromethyl, - N (R _a) (R _b), -OR _a , -SR _a , -C (0) R _a , -C (0) OR _a , -C (0) N (R _a ) (R _b ), -OC (0) R _a ; wherein each R _a and R _b is independently selected from hydrogen, Ci-C ₆ alkyl, aryl, heterocycle, heteroaryl and trifluoromethyl. The compounds of the present invention may be in the form of salts, preferably pharmaceutically acceptable salts, or in the form of solvates. The term "pharmaceutically acceptable salts" refers to any salt that upon administration to the recipient can provide (directly or indirectly) a compound as described herein. The term "solvate" according to this invention is understood to mean any form of the active compound according to the invention that has another molecule (most likely a polar solvent) attached to it by non-covalent bonds. Examples of solvate sites include hydrates and alcoholates, eg methanolate. Preferably, the solvates are pharmaceutically acceptable solvates. The preparation of salts and solvates can be carried out by methods known in the art. Examples of acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, iohydrate, sulfate, nitrate, phosphate and organic acid addition salts, such as, for example, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate and p-toluenesulfonate. Preferably, trifluoroacetate.

Pharmaceutical compositions According to a further aspect, the invention relates to a pharmaceutical composition comprising at least one compound of the invention, as previously defined, and at least one pharmaceutically acceptable excipient.

The term "pharmaceutically acceptable" refers to molecular compositions and entities that are physiologically tolerable and normally do not produce an allergic reaction or a similar unfavorable reaction such as gastric disorders, dizziness and the like, when administered to a human being or an animal. Preferably, the term "pharmaceutically acceptable" means that it is approved by a regulatory agency of a state or federal government or is included in the US pharmacopoeia or other pharmacopoeia generally recognized for use in animals, and more particularly in humans.

The term "excipient" refers to a diluent, adjuvant, carrier or vehicle with which the active substance is administered. Such pharmaceutical excipients may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Preferably water or saline solutions of aqueous solution and aqueous solutions of dextrose and glycerol are used as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. For a better formulation of the present invention, dispersing agents, such as surfactants and / or polymers, can also be added.

The pharmaceutical composition of the invention can be administered in a plurality of pharmaceutical forms of administration, for example solid (tablets, pills, capsules, granules, etc.) or liquid (solutions, suspensions or emulsions). Administration may be carried out, for example, orally (sublingually, gastroenterically, rectally), parenterally (intravenously, intraarterially, intracardiacly, subcutaneously, transdermally, intraperitoneally or intramuscularly) or topically.

Applications

The interaction between small molecules and double stranded DNA causes changes in the structure and functionality of said DNA, which may allow modulating, by activation or inhibition, various processes in which DNA is involved, such as transcription or replication, and thereby regulate cellular activity.

As described in this application, the use of photolabile protecting groups makes it possible to temporarily inactivate small double stranded DNA binding molecules. Subsequent irradiation with light allows the molecule to be released again and, thus, spatially and temporarily control the activity of the molecule, which could allow control over biological processes such as gene expression, protein synthesis and cell proliferation.

In addition, the spectral characteristics of these small molecules change when they bind to DNA, so those that absorb in UV are usually used as markers or dyes of DNA and cells (eg acridine orange, DAPI, ethidium bromide, propinium iodide Hoechst 33258 and Hoechst 33342). Again, following a protection / deprotection process (inactivation / activation) as previously mentioned, it is possible to effectively control the activity of these markers. Therefore, a further aspect is directed to the use of the compounds of the invention, as previously described, to mark in vitro DNA sequences or cells.

In a further aspect, the invention is directed to an in vitro method for marking DNA sequences or cells, comprising (a) interacting the DNA sequence or the cell with a small double stranded DNA binding molecule, and ( b) irradiate the sample with UV light.

The changes caused by the union of small molecules to double-stranded DNA cause functional alterations in the DNA due to the impossibility, decrease or modification of the interaction of other molecules with DNA, such as transcription factors, enzymes, etc. Generally, these small double-stranded DNA binding molecules act by inhibiting the processes of DNA transcription, replication and repair, which inhibits cell reproduction and causes death. Therefore, these types of molecules have important therapeutic applications as antibacterial, antiviral, antifungal, antiparasitic and antitumor agents. A further aspect refers to the use of the compounds of the invention, as previously described, for the preparation of a medicament. Another additional aspect relates to the use of the compounds of the invention, as previously described, for the preparation of a medicament for the treatment of proliferative diseases and microbial diseases.

In another aspect, the invention is directed to a therapeutic method, which comprises the administration of the compounds of the invention and subsequent irradiation with UV light. Preferably, the therapeutic method is directed to the treatment of proliferative diseases and microbial diseases.

The term "proliferative diseases" refers to abnormal cell growth or abnormal cell growth without physiological control. Proliferative diseases can be benign or malignant tumors (cancers). Non-cancerous proliferative diseases include lipomas, adenomas, hemangiomas, lymphanginomas, nevus, teratomas, fibromas, myxomas, chondromas, osteomas, meningiomas, gliómicos tumors, leiomyomas, rhabdomyomas, papillomas, angiomas and myomas. Examples of cancers include, but are not limited to, spleen, colorectal and / or colon cancer, colon carcinomas, ovarian carcinomas, ovarian cancer, breast cancer, uterine carcinomas, lung cancer, stomach cancer, cancer of the esophagus, liver cancer, carcinomas of the pancreas, kidney cancer, bladder cancer, prostate cancer, testicular cancer, bone cancer, skin cancer, sarcoma, Kaposi sarcomas, brain tumors, myosarcomas, neuroblastomas, lymphomas and leukemia, melanoma, glioma, medulloblastoma, head and neck carcinoma.

In a particular embodiment, the proliferative disease is cancer.

The term "microbial diseases" as used herein includes diseases caused by bacteria, viruses, fungi and parasites. Therefore, a particular embodiment refers to the use of the compounds of the invention, as previously defined, in the preparation of a medicament for the treatment of microbial diseases, preferably bacterial, viral, fungal and parasitic diseases.

The compounds of the present invention can be prepared by reacting the small double stranded DNA binding molecule and the photlabile protecting group, by synthetic methods common in the state of the art and known to the subject matter expert (eg PJ Kocienski, "Protecting Groups", Thieme 2005; "March 's Advanced Organic Chemistry", Wiley-Interscience 2001; TW Greene, "Protective Groups in Organic Chemistry", Wiley 2007).

The invention will be illustrated below by tests performed by the inventors.

EXAMPLES

 Example 1. Method of synthesis of 2,2 '- {L5-pentanediylbis [imino-4, l-phenylene (iminomethylene) iminol | bis [(2-nitrophenyl) acetic acid (Nmoc ^ -pentamidine, © 1)

Synthesis of (([(2,5-dioxo-l-pyrrolidinyl) oxylcarbonyl | oxy) (2-nitrophenyl) terebutyl acetate (7)

On a solution of alcohol 6 (200 mg, 0.79 mmol - obtained according to the procedure described by Rossi et al. J. Biol. Chem. 1997, 272, 32933-32939) dissolved in MeCN (8 mL) Et ₃ N ( 320 mg, 3.16 mmol) and Ν, Ν ^' -disuccinimidyl carbonate (213 mg, 0.95 mmol). It was stirred for 8 h at room temperature under Ar atmosphere, the solvent was removed and the residue was purified by silica gel chromatography (60% AcOEt / hexanes), yielding the product as a white solid (178 mg, 60%).

1H-NMR (250MHz, CDC1 ₃ ): 1.12-1.43 (m, 9H), 2.83 (s, 4H), 6.7 (s, 1H), 7.55-

7.62 (m, 1H), 7.5-7.5 (m, 2H), 7.9-8.1 (m, 1H).

¹³ C-NMR (250MHz, CDC1 ₃ ): 25.4 (CH2), 27.6 (CH3), 75.7 (CH), 84.8 (C), 125.4 (CH), 128.0 (C), 128.8 (CH), 130.3 (CH) , 133.9 (CH), 147.6 (C), 150.8 (C), 164.3 (C), 168.2 (C).

Synthesis of 2,2 '- {L5-pentanediylbis [imino-4, l-phenylene (iminomethylene) iminoll bis [(2-nitrophenyl) acetic acid (Nmoc ^ -pentamidine, © 1) The trifluoroacetic di-salt of azapentamidine (50 mg, 0.088 - obtained according to the procedure described by Bakunova et al. J. Med. Chem. 2009, 52, 2016-2035) was dissolved in 1.8 mL of a previously prepared DIEA solution / DMF (0.195 M). The compound ({[(2,5-dioxo-l-pyrrolidinyl) oxy] carbonyl} oxy) (2-nitrophenyl) tert-butyl acetate (75 mg, 0.19 mmol) was added to the solution and the resulting reaction crude it was stirred magnetically under Ar's atmosphere and in the absence of light for 8 hours. After that time the solvent was removed under reduced pressure and the product was isolated and purified by silica gel column chromatography, obtaining as a yellowish solid.

The isolated product containing the tere-butyl protecting group was dissolved in 4.5 mL of CH ₂ CI ₂ and cooled to 0 ° C. Then, 4.4 mL of TFA was added slowly. It was allowed to react with magnetic stirring for 2 h and subsequently the solvent was removed under reduced pressure. Finally, the reaction crude was purified in the reverse phase (Water / MeCN) and the fractions containing the desired product were lyophilized to obtain a yellowish solid (63 mg, 0.079 mmol, 89% in total).

1 H NMR (300 MHz, MeOD-d ₄ ): 1.28-1.30 (m, 2H), 1.69-1.73 (m, 4H), 2.86 (s, 2H, H), 2.99 (s, 2H, H), 3.24 ( t, j = 6.95 Hz, 4H), 6.74 (d, J = 9.12 Hz, 4H), 6.99 (s, 2H, CH), 7.67-7.83 (m, 12 H), 8.12 (s, 1H, NH), 8.14 (s, 1H, NH).

¹³ C NMR (300 MHz, MeOD-d ₄ ): 25.6 (CH ₂ ), 29.7 (CH ₂ ), 43.7 (CH ₂ ), 73.5 (CH), 111.8 (C), 112.9 (CH), 126.4 (CH) , 129.7 (C), 131.0 (CH), 131.8 (CH), 132.4 (CH), 135.0 (CH), 149.7 (C), 154.0 (C), 157.0 (C), 165.5 (C), 169.7 (C) .

Example 2. Method of synthesis of 3,8-bis (([(4,5-dimethoxy-2- nitrobenzyl) oxylcarbonyl | amino) -5-methyl-6-phenylphenanthridinium (Nvoc ^ -etidium, © 2)

 To a solution of ethidium bromide (100 mg, 0.253 mmol) in 25 mL of

DIEA / DMF (0.195 M) was added moderately nitroveratril chloride (209 mg, 0.759 mmol). The resulting mixture was allowed to react with magnetic stirring, under Argon atmosphere and in the absence of light for 16 hours. After concentrating the solvent under reduced pressure, the reaction crude was purified in reverse phase. (Water / MeCN) and the fractions containing the desired product were lyophilized to obtain the product as an orange trifluoroacetic salt (77 mg, 0.086 mmol, 34%).

Example 3. Synthesis procedure of 4,5-dimethoxy-2-nitrobenzyl [(4- {6 - [({[(4,5-dimethoxy-2-nitrobenzyl) oxyl carbonyl I amino) (imino) metill-1 H -indole-2-yl | phenyl) (imino) methylcarbamate (Nvoc _r DAPL © 3)

DAPI (12 mg, 0.034 mmol) was dissolved in 0.7 mL of DMSO in an eppendorf. Then, Et ₃ N (30 mg, 41 μΐ., 0.296 mmol, 8 equiv) and finally nitroveratril chloride (56 mg, 0.204 mmol, 6 equiv) was added. The resulting mixture was allowed to react with magnetic stirring and in the absence of light for 16 hours. The reaction crude was purified in reverse phase (Water / MeCN) and the fractions containing the desired product were lyophilized to obtain the product as a yellowish solid (14 mg, 0.018 mmol, 54%).

1H NMR (500 MHz, MeOH-d ₄ ): 3.93 (s, 3H), 3.94 (s, 3H), 3.99 (s, 3H), 4.00 (s, 3H), 5.76 (s, 2H), 5.78 (s , 2H), 7.27 (s, 1H), 7.33 (d, J = 4.56 Hz, 2H), 7.51 (dd, J = 8.43, 1.72 Hz, 1H), 7.81 (d, J = 2.99 Hz, 2H), 7.87 (d, J = 8.44 Hz, 1H), 7.97 (d, J = 8.57 Hz, 2H), 8.03 (s, 1H), 8.16 (d, J = 8.49 Hz, 2H).

¹³ C NMR (300 MHz, MeOH-d ₄ ): 56.9 (CH ₃ ), 57.1 (CH ₃ ), 67.4 (CH ₂ , Bn), 103.3 (CH), 109.5 (CH), 113.0 (CH), 114.3 ( CH), 120.6 (CH), 121.8 (C), 122.9 (CH), 126.0 (C), 126.2 (C), 127.3 (CH), 130.7 (CH), 135.4 (C), 138.3 (C), 138.7 ( C), 141.6 (C), 142.4 (C), 150.5 (C), 154.6 (C), 155.2 (C), 167.9 (C), 168.7 (C).

Example 4. Synthesis procedure of 2,2 '- {L5-pentanediylbis [imino-4, l-phenylene (iminomethylene) iminol | bis (([(2,5-dioxo-1-pyrrolidiniDoxil carbonyl I oxy) (4,5-dimethoxy-2-nitrophenyl) l (Nvoc ^ -pentamidine, © 4)

Synthesis of ({[(2,5-dioxo-l-piirolidinyl) oxylcarbonyl | oxy) -4,5-dimethoxy-2-nitrophenyl (9)

On a solution of the alcohol 8 (500 mg, 2.35 mmol) dissolved in MeCN (23.5 mL) was added Et ₃ N (950 mg, 9.4 mmol) and Ν, Ν ^' -disuccinimidyl carbonate (721 mg, 2.82 mmol). It was stirred for 8 h at room temperature under Ar atmosphere, the solvent was removed and the residue was purified by silica gel chromatography (60% AcOEt / hexanes), yielding the product as a yellow solid (607 mg, 73%).

1H-NMR (250MHz, CDC1 ₃ ): 2.85 (s, 4H), 3.96 (s, 3H), 4.05 (s, 3H), 5.78 (s, 2H), 7.04 (s, 1H), 7.75 (s, 1H ).

¹³ C-NMR (250MHz, CDC1 ₃ ): 25.4 (CH2), 56.4 (CH3), 56.6 (CH3), 69.1 (CH2), 108.2 (CH), 108.6 (CH), 125.3 (C), 139.1 (C) , 148.5 (C), 151.3 (C), 154.1 (C), 168.4 (C).

 Synthesis of 2,2 '- {L5-pentanediylbis [imino-4J-phenylene (iminomethylene) iminollbis (([(2,5-dioxo-l-pyrrolidinyl) oxylcarbonyl | oxy) (4,5-dimethoxy-2-nitrophenyl) l (Nvoc ^ -pentamidine, © 4)

 The trifluoroacetic di-salt of azapentamidine (50 mg, 0.088 - obtained according to the procedure described by Bakunova et al. J. Med. Chem. 2009, 52, 2016-2035) was dissolved in 1.8 mL of a previously prepared DIEA solution / DMF (0.195 M). Compound 9 (93 mg, 0.26 mmol) was added to the solution and the resulting reaction crude was stirred magnetically under Ar's atmosphere and in the absence of light overnight. After that time the solvent was removed under reduced pressure and the product was isolated and purified by silica gel column chromatography, obtaining the desired product (60 mg, 0.073 mmol, 83%).

1 H NMR (300 MHz, DMSO-d ₆ ): 1.46 (m, 2H), 1.59 (m, 4H), 3.08 (dd, J =

12.42, 6.59 Hz, 4H), 3.87 (s, 6H), 3.87 (s, 6H), 5.37 (s, 4H), 6.42 (t, J = 5.35 Hz, 2H), 6.57 (d, J = 8.97 Hz, 4H), 7.21 (s, 2H), 7.69 (s, 2H), 7.81 (d, J = 8.88 Hz, 4H).

¹³ C NMR (300 MHz, DMSO-d ₆ ): 24.1 (CH ₂ ), 28.2 (CH ₂ ), 42.2 (CH ₂ ), 55.9 (CH ₃ , OMe), 56.0 (CH ₃ , OMe), 62.8 (CH2 ), 108.0 (CH), 110.5 (CH), 111.1 (CH), 119.2 (CH), 127.7 (C), 129.3 (CH), 139.6 (C), 147.6 (C), 152.3 (C), 153.0 (C ), 163.3 (C), 166.8 (C). Fluorescence Spectroscopy

 The measurements were made on a Jobin-Yvon Fluoromax-3 (DataMax 2.20) device coupled to a Wavelength Electronics LFI-3751 temperature controller. All data was measured at 20 ° C.

 In vitro experiments with © 1 were performed with the following parameters: increase: 1.0 nm; integration time: 0.2 s; excitation groove width: 2.0 nm; emission slot width: 4.0 nm; excitation wavelength 329 nm and measured from 345 nm to 500 nm.

 In vitro experiments with © 2 were performed with the following parameters: increase: 1.0 nm; integration time: 0.25 s; excitation groove width: 2.0 nm; emission slot width: 4.0 nm; excitation wavelength 360 nm and measured from 380 nm to 600 nm.

 The hairpin oligonucleotide containing the AAATTT target sequence (SEQ ID NO: 1) was supplied by Thermo Fischer Scientific GmbH.

 The deprotection of the photolabile protecting groups was carried out by irradiation with a transilluminator with an 8 watt lamp (max. Approx. 360 nm).

Example 5. In vivo tests with protected compounds © 2 and © 3

 The cells in which the experiments were performed in vivo belong to the Vero cell line, the culture medium of these is DMEM buffer (Dulbecco Modified Eagle Medium) containing 10% FBS (Fetal Bovine Serum). The day before the experiment, they were transferred to wells containing their corresponding 15 mm coverslips, then washed and stored with serum-free PBS.

The tests with the compounds (Nvoc ₂ -ethidium, © 2) and (Nvoc ₂ -DAPI, © 3) were carried out at a concentration of 50 microM except in the case where the effect of the concentration in relation to its permeability was studied to cell membranes.

Once added to the culture medium (Nvoc ₂ -ethidium, © 2) or (Nvoc ₂ -DAPI,

© 3), the cells were incubated for 30 min at room temperature and in the dark, after that time they were washed 3 times with PBS, once washed the coverslips were transferred with the cells to the slides where they were mounted with Mowiol 4-88 ® [100 mg / mL in 100 mM Tris-HCl pH 8.5, 25% glycerol and 0.1% DABCO (as an antifungal agenta)] for irradiation and subsequent visualization with fluorescence microscopy. The deprotection was performed with a UV transilluminator for gels during different times before being observed in the fluorescence microscope. The selected images were taken with an Olympus DP-50 digital camera, then these were processed with the Adobe Photoshop (Adobe Systems) software.

 Figures 5 to 8 show the images taken by fluorescence microscopy of the cells incubated with compounds 2, © 2, 3 and © 3, before and after being irradiated with UV light.

Example 6. Disk diffusion tests

 The antimicrobial activity of compound © 4 was measured by the conventional method of disk diffusion with Candida albicans, following the procedure of the Clinical and Laboratory Standards Institute (CLSI, 2006).

 The antimicrobial solution will be prepared using water or DMSO as solvent. The sterile 6 mm diameter discs (Liofilchem, Italy) embedded in the drug were kept on the surface of Mueller Hinton Agar (Cultimed, Spain) inoculated with the yeast.

 The plates were incubated at 37 ° C for 24 h and the zones of inhibition were measured in millimeters. The results obtained are shown in Figure 9, where the antimicrobial activity of compound © 4 is shown after being irradiated with UV light.

Claims

1. A compound formed by a small double stranded DNA binding molecule that is covalently bound to a photolabile protecting group, provided that said compound is not in turn a double stranded DNA binding molecule. 2. A compound according to claim 1, wherein the small double stranded DNA binding molecule is selected from an intercalating agent, an alkylating agent or a minor groove binding molecule. 3. A compound according to any of the preceding claims, wherein the small double stranded DNA binding molecule is a DNA dye, an antitumor agent or an antimicrobial agent. 4. Compound according to any one of the preceding claims, wherein the small double stranded DNA binding molecule is a compound of formula (la) or (Ib):

where  A and B are each independently selected from optionally substituted aryl and heteroaryl; X may not exist, so that A and B are condensed, or it represents a bond, -O-, -S-, -NH-, -OYO-, -SYS-, -NH-Y-NH-, or a optionally substituted Ci-C ₆ alkyl, aryl or heteroaryl; Y is selected from optionally substituted Ci-C ₈ alkyl, aryl and heteroaryl, or a salt or solvate thereof. 5. Compound according to any of the preceding claims, wherein the small double stranded DNA binding molecule is selected from pentamidine, azapentamidine, ethidium bromide and 4 ', 6-diamidinium-2-phenylindole. Compound according to any of the preceding claims, wherein the photolabile protector is a compound of formula (II):

where n is selected from 0, 1, 2, 3 and 4; R ¹ and each R ² are independently selected from hydrogen, Ci-C ₆ alkyl, Ci-C ₆ haloalkyl, C ₆ -C ₅ aryl, C ₃ -C 15 heteroaryl and optionally substituted C ₃ -C ₁₅ heterocycle, N ₂ , CN, halogen, - OR ', -SR', -S (0) R ', -S (0) ₂ R', - OS (0) ₂ R ', -N (R') (R "), -C (0) R ', -C (0) OR', -C (0) N (R ') (R"), -OC (0) R 'and - N (R') C (0) R "; wherein each R 'and R" is independently selected from hydrogen, Ci-C ₆ haloalkyl _Ci-C6, C3-C7 , optionally substituted C ₆ -Ci5 aryl, C3-C15 heteroaryl and C3-C15 heterocycle; or two R ² groups form, together with the phenyl ring to which they are attached, a heterocycle group. Compound according to any of the preceding claims wherein: n is selected from 0, 1 and 2; R ¹ is selected from hydrogen, methyl, trifluoromethyl and carboxyl; R ² represents methoxy, or two adjacent R ² radicals form a group -0-CH ₂ -0-. Compound according to any of the preceding claims, wherein at least one of R ¹ and R ² comprises an ionizable group at physiological pH. Compound according to the preceding claim, wherein at least one of R ¹ and R ² is a carboxyl group. Compound according to any of the preceding claims wherein the photolabile protector is selected from:

11. Compound according to any of the preceding claims selected from a compound of formula (Illa) and (Illb):

 (Illb) wherein A, B, X, n, R ¹ and R ² are as defined in the preceding claims. Compound according to any of the preceding claims selected from:

or a salt or solvate thereof. Use of a compound as defined in any one of claims 1 to 12 for in vitro control of gene expression and protein synthesis. 14. Use of a compound as defined in which one of claims 1 to 12 for in vitro labeling of DNA sequences or cells. 15. Pharmaceutical composition comprising at least one compound as defined in any of claims 1 to 12, and at least one pharmaceutically acceptable excipient. 16. Use of a compound as defined in any of claims 1 to 12, to prepare a medicament. 17. Use of a compound as defined in any of claims 1 to 12, for the preparation of a medicament for the treatment of proliferative diseases or microbial diseases. 18. Use according to claim 17, wherein the proliferative disease is cancer. 19. Use according to claim 17, wherein the microbial disease is a bacterial, viral, fungal or parasitic disease.